QPCRBIO Probe Mixes

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.

Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).

The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:

1. Determine the correct ROX concentration to optimise real-time PCR results, and
2. Check the ROX settings on the software used to set up the reaction

A useful selection tool for the most commonly used systems can be found here.

qPCRBIO Probe Mixes are ready to use qPCR 2x Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence.

The qPCRBIO Probe Mixes that contain passive reference dyes come in different formulations, each with a different concentration of the passive reference dye:

• qPCRBIO Probe Mix Lo-ROX (PB20.21) contains 112 nM ROX.
• qPCRBIO Probe Mix Hi-ROX (PB20.22) contains 1.12 µM ROX.
• qPCRBIO Probe Mix No-ROX (PB20.23) does not contain ROX.
• qPCRBIO Probe Mix Separate-ROX (PB20.24) 2x mix contains no ROX and include a separate tube of 50 µM ROX additive. This enables you to choose what concentration of ROX you’d like to use.

You can use our qPCR Selection Tool under the Resources drop-down menu to determine which of our mixes are best suited for your qPCR machine.

Yes, PCR products generated with qPCRBIO Probe Mixes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-d(A)-tailed and may be used for TA cloning or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, we recommend purifying the PCR products using any standard PCR clean-up kit.

All qPCRBIO Probe Mixes contain MgCl2 at a concentration of 12 mM. This means the final concentration in the reaction is 6 mM.

All qPCRBIO Probe Mixes contain dNTPs at a concentration of 2 mM (0.5 mM each). This means the final concentration in the reaction is 1 mM (0.25 mM each).

Higher Ct values are generally indicative of delayed amplification. This could most likely be due to an excess of template in the reaction resulting in the primers and probes being bound on different DNA molecules. Samples usually have a lot of DNA other than the target gene and this can scatter the oligos. We recommend diluting the samples (10x-1000x) to solve this.

Moreover, annealing/extension temperature can also be increased to make the binding of the oligos more specific to the target sequence and decrease background signal.

It has been reported that efficiency can decrease with subsequent dilutions for the standard curve. We recommend avoiding this by diluting the standards in 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.05% Tween-20. EDTA is a chelating agent and it plays a role in preventing DNAse activity1. Tween-20 is a detergent and prevents the DNA from adsorbing to the sides of the tubes2. Most microcentrifuges are made of polypropylene and research has demonstrated that DNA sticks very well to polypropylene3.

Standards should not be frozen after diluting them. Even in the presence of detergent, freezing seems to cause DNA to bind irreversibly to polypropylene. We suggest leaving your standards at 4°C and preparing a fresh batch every few weeks.

1  Barra, G. B. et al. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin Biochem 48, 976-981, doi:10.1016/j.clinbiochem.2015.02.014 (2015).

2  Linnarsson, S. Recent advances in DNA sequencing methods – general principles of sample preparation. Exp Cell Res 316, 1339-1343, doi:10.1016/j.yexcr.2010.02.036 (2010).

3  Gaillard, C. & Strauss, F. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments. Technical Tips Online 3, 3 (1998).

Ensure you are using the right concentration of ROX because different instruments require different ROX concentrations. For example, if a qPCRBIO Probe Mix Hi-ROX is used with an instrument requiring a Lo-ROX mix, the software will normalise the qPCRBIO signal against the Hi-ROX level. This will significantly reduce the fluorescence level of the qPCRBIO mix relative to competitors’ mixes, if in that case the Lo-ROX mix was chosen for the competitor.

When comparing mixes from different manufacturers, it is better to carry out separate runs or turn the passive reference off before analysing data.

It’s most likely because the time for the 1st step (hot start) is too short. Ensure that the hot start phase is done at 95°C for 2 minutes to fully activate the enzyme. The recommended thermal profile is:

• 95°C (120 seconds)
• 40 cycles: 95°C (5-15 seconds) – 60°C (20-30 seconds)
• Melt

If non-specific products are still obtained, we recommend raising the annealing/extension temperature from 60°C to 65°C, depending on the primer set used.

Yes, this storage buffer is compatible. The EDTA will chelate some of the magnesium in the mix, but not significantly enough to affect the reaction.

If inhibition is observed, the amount of template in the reaction can be decreased. This will increase the Ct value but lower the likelihood of inhibitors interfering with the Taq DNA polymerase activity. If this doesn’t work, try adding 0.4-4 mg/ml of BSA to the reaction1,2. Ensure the cycling conditions in our product manual are adhered to.

 1  Kreader, C. A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62, 1102-1106 (1996).

2  Wilson, I. G. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63, 3741-3751 (1997).

Yes. The usual activation time of 2 minutes is not a long enough time to ensure release of the DNA from the bacteria or virus sample. The activation step can be increased to 15 minutes without affecting the reaction yield.

We recommend using 0.4 µM of each primer. There is a degree of flexibility around this recommended concentration however, the primer concentration should not be increased, as this may significantly affect the activity of the enzyme.

Different products could give a different plateau of fluorescence. However, this has no impact on quantification accuracy and Ct values will not differ among products.

There are different options to consider when optimising the reaction:

• Reduce the annealing/extension time to 5 seconds
• Increase the annealing/extension temperature from 60 to 65°C
• Dilute the DNA template by starting with 5ng of DNA and using a 10x template dilution series. In addition to running these on a gel to see if the non-specific products persist, the efficiency of the reaction can be calculated with the software of the qPCR instrument after doing the template dilution. If the efficiency is between 90 – 110%, then the amplicon is being doubled every cycle.

No. Apart from ROX, there is no other dye in our mixes. You can therefore use any fluorophore-conjugated probe for your reaction. Keep in mind that if you’re using qPCRBIO Probe Blue Mixes, refer to Table 1 in the respective product manuals to ensure the blue dye will not interfere with the fluorescence of your fluorophore.